CN113332490A - Bone repair support and preparation method thereof - Google Patents

Abstract

The invention discloses a bone repair scaffold and a preparation method thereof, wherein the bone repair scaffold takes tin-doped calcium phosphate as a main raw material and is formed by 3D printing, and the doping amount of tin is 1-5 wt%. The doped tin is utilized to improve the mechanical property of the existing beta-TCP bracket, the tin-doped beta-TCP bracket with good biocompatibility is prepared, and the doping of the tin improves the capability of the bracket in inducing the proliferation and differentiation of the mesenchymal stem cells, thereby being beneficial to the application of the bracket in bone defect repair.

Description

Bone repair support and preparation method thereof

Technical Field

The invention belongs to the technical field of medical material preparation, and particularly relates to a bone repair stent and a preparation method thereof.

Background

3D printing (3 DP) is also called additive manufacturing (additive manufacturing), is carried out at room temperature or body temperature, can quickly and accurately prepare materials, shortens the research and development time of products and savesThe production cost is a good choice for preparing artificial organs or substitute materials. At present, the common materials used in the aspect of bone tissue engineering by 3D printing are bioceramic scaffold materials such as Hydroxyapatite (HA), tricalcium phosphate (TCP), calcium silicate (CaSiO)₃) And composite scaffolds of these and organic polymers.

Tricalcium phosphate (TCP) of formula Ca₃(PO₄)₂The beta-TCP has similar components with natural bones, has good biocompatibility, biodegradability and osteoinductivity, can be directly fused with bones when being implanted into bone tissues, does not generate rejection, inflammation and toxic or side effect, and can promote the regeneration of new bone tissues. After the beta-TCP material enters the body, body fluid can enter the pores of the beta-TCP material to dissolve the crystal grains thereof, and simultaneously, under the erosion of the body fluid, the beta-TCP material can release bioactive Ca²⁺And PO₄ ^3-These ions can be ion exchanged with body fluids, and thus the specific surface area, crystallinity and pH of the β -TCP material can play a critical role in its degradation rate. In addition, macrophages and osteoclasts in the body can phagocytose the beta-TCP material and also degrade the material. beta-TCP is strong in bending strength and fracture toughness, but weak in self-alkalinity, poor in mechanical property and poor in casting formability, and is lower than the mechanical property of human cortical bone, so that the beta-TCP is not used as a bone repair material alone, and needs to be compounded with other substances to improve the mechanical property. At present, the beta-TCP ceramic material is compounded with other materials with better strength to prepare a two-phase and multi-phase composite material, which is an effective method for improving the mechanical strength, regulating the degradation rate and increasing the biological activity.

Tin is one of the trace elements necessary for human life activities. It has important influence on various physiological activities of human body and human health maintenance. Tin and its compounds are poorly absorbed and accumulated in human tissues and are rapidly excreted mainly through the kidneys, thus being good in biocompatibility. It has been found that tin improves the castability and corrosion resistance of the alloy material. In the field of bone repair and biomaterials, there are few reports on tin, and further research is needed.

Disclosure of Invention

The invention aims to provide a bone repair bracket and a preparation method thereof, and the calcium phosphate bone repair bracket doped with tin is prepared by a 3D printing technology. The doping of tin can improve the mechanical property of the calcium phosphate scaffold and the capability of inducing osteogenic differentiation of bone marrow mesenchymal stem cells, has good biocompatibility and is beneficial to the application of the calcium phosphate scaffold in bone defect repair.

In order to achieve the purpose, the invention adopts the following technical scheme:

a bone repair scaffold is prepared from tin-doped calcium phosphate as main raw material by 3D printing, wherein the tin doping amount is 1-5 wt%.

The preparation method comprises the following steps:

(1) designing a support structure: the cylindrical shape has the bottom area of 8mm and the height of 4 mm; the Simplify3D software parameters are set as: the printing speed is 4mm/s, and the filling rate is 40 percent;

(2) preparation of printing ink: sieving the beta-TCP powder by a 200-mesh sieve, and uniformly mixing the beta-TCP powder with tin powder to obtain Sn @ beta-TCP mixed powder with the metal tin content of 1-5%; adding the frozen section embedding agent according to the mass ratio of 1:1, and mixing and stirring for 10min to obtain printing ink;

(3) and printing the printing ink into a support through an extrusion type 3D printer, drying at room temperature for 48h, and putting the support into a muffle furnace for high-temperature sintering to obtain the bone repair support.

The frozen section embedding medium is aqueous solution of polyethylene glycol and polyvinyl alcohol.

The high-temperature sintering is carried out at a heating speed of 10 ℃/min, and the temperature is raised to 700-.

The invention has the beneficial effects that: the doped tin is utilized to improve the mechanical property of the existing beta-TCP bracket, the tin-doped beta-TCP bracket with good biocompatibility is prepared, and the doping of the tin improves the capability of the bracket in inducing the proliferation and differentiation of the mesenchymal stem cells, thereby being beneficial to the application of the bracket in bone defect repair.

Drawings

In FIG. 1, a is a schematic diagram of a 3D printer, b is a bracket photo, and c is a scanning electron microscope image of a bracket (beta-TCP, beta-TCP @ 1% Sn, beta-TCP @ 3% Sn, beta-TCP @ 5% Sn).

FIG. 2 shows XRD (FIG. 2a) and XPS (FIG. 2b) of β -TCP @ 5% Sn scaffolds prepared at different sintering temperatures (700 ℃, 900 ℃, 1100 ℃).

Fig. 3 is a mechanical property characterization of the stent for different tin doping amounts.

Fig. 4 is biocompatibility data for stents with different tin doping levels.

Fig. 5 is a graph showing the effect of scaffolds with different tin doping amounts on the osteogenic differentiation capacity of BMSC cells.

FIGS. 6 and 7 show the effect of scaffolds with different tin doping amounts on BMSC cell gene expression profiles.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

Example 1: 3D printing preparation of bone repair bracket of tin-doped calcium phosphate

1. The support structure to be printed is designed using the software Soildworks sorfware (2014 version). The support is designed into a cylindrical structure, the bottom area is 8mm, and the height is 4 mm. The Simplify3D software parameters are set as: the printing speed was 4mm/s and the filling rate was 40%.

2. Preparation of printing ink: selecting a commercial frozen section embedding medium (a water-soluble mixture of polyethylene glycol and polyvinyl alcohol) as a binder, taking metallic tin powder and beta-TCP powder as raw materials, and preparing and printing four parts of Sn @ beta-TCP slurry with the metallic tin content of 0%, 1%, 3% and 5% respectively according to a ratio to serve as printing ink. The method comprises the following specific steps:

(1) sieving the beta-TCP powder by a 200-mesh sieve to obtain beta-TCP powder with uniform size;

(2) the formulation is as shown in Table 1:

TABLE 1 slurry proportioning Table

And (3) blending Sn @ beta-TCP mixed powder with the metal tin contents of 0, 1%, 3% and 5% according to a slurry proportioning table.

(3) The mixed powder and the frozen section embedding medium are mixed and stirred for 10min at room temperature in a ratio of 1: 1.

3. And (3) drying the prepared printing ink at room temperature for 48h after printing the support by an extrusion type 3D printer, and putting the support into a muffle furnace for high-temperature sintering. The sintering conditions were set at a heating rate of 10 ℃/min, and the temperature was raised to 1100 ℃ for 3 hours.

Fig. 1a is a schematic diagram of a 3D printer. Figure 1b is a photograph of the stent size printed. It can be seen that the diameter of all stents after sintering is significantly smaller than that before sintering. FIG. 1c shows the morphology of several scaffolds (. beta. -TCP,. beta. -TCP @ 1% Sn,. beta. -TCP @ 3% Sn,. beta. -TCP @ 5% Sn) as observed by scanning electron microscopy (SEM, Sigma 300, Zeiss, Germany). The results show that the scaffold exhibits a porous structure consisting of a plurality of particles, with a larger pore size after sintering than before sintering. As the doping level of tin increases, more and more particles change from round to angular polygonal.

Example 2: and (3) carrying out X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) characterization on beta-TCP @ 5% Sn prepared at different sintering temperatures, and optimizing the sintering temperature.

3D printing preparation of beta-TCP @ 5% Sn at different sintering temperatures:

1. the support structure to be printed is designed using the software Soildworks sorfware (2014 version). The support is designed into a cylindrical structure, the bottom area is 8mm, and the height is 4 mm. The Simplify3D software parameters are set as: the printing speed was 4mm/s and the filling rate was 40%.

2. Preparation of printing ink: tin was mixed with beta-TCP at 5% doping ratio, and further mixed with 3g of binder and stirred for 10 min.

3. And (5) drying the support at room temperature for 48h after the support is printed by using a 3D printer, and placing the support into a muffle furnace for high-temperature sintering. The sintering conditions were set at a heating rate of 10 ℃/min, and the temperature was raised to 700 ℃, 900 ℃ and 1100 ℃ for 3 hours, respectively.

As shown in FIG. 2a, when the composition of SnO2 in the beta-TCP @ 5% Sn scaffold prepared at different sintering temperatures (700 ℃, 900 ℃ and 1100 ℃) was analyzed by X-ray diffraction (XRD), it could be seen that the characteristic peak of SnO2 in the beta-TCP @ 5% Sn scaffold prepared at the sintering temperature of 1100 ℃ is more distinct than that of the beta-TCP @ 5% Sn scaffold prepared at the sintering temperatures of 700 ℃ and 900 ℃. FIG. 2b shows that the oxidation level of Sn element in beta-TCP @ 5% Sn scaffolds prepared at different sintering temperatures (700 deg.C, 900 deg.C, 1100 deg.C) is analyzed by X-ray photoelectron diffraction spectrum, and the peak of Sn3d is most clear when sintering at 1100 deg.C. The above results all show that sintering at 1100 ℃ is the optimum sintering temperature for the material.

Example 3: investigating the mechanical property of Sn @ TCP by a universal material testing machine

Scaffolds of beta-TCP, beta-TCP @ 1% Sn, beta-TCP @ 3% Sn, beta-TCP @ 5% Sn were prepared as in example 1. And (4) inspecting the mechanical property of the prepared bracket by using a universal material testing machine. The best mechanical properties of the beta-TCP @ 5% Sn scaffold compared with other scaffolds (beta-TCP, beta-TCP @ 1% Sn, beta-TCP @ 3% Sn) can be seen through the compressive strength, the compressive modulus, the compressive yield force and the compressive stress curve (figures 3 a-d). The doping of tin is helpful for improving the mechanical property of the beta-TCP support, and the larger the doping amount is, the more the mechanical property is improved.

Example 4: sn @ TCP stent biocompatibility experiment

1. Cell proliferation detection kit

Preparation of β -TCP, 1% Sn @ β -TCP, 3% Sn @ β -TCP, 5% Sn @ β -TCP scaffolds 48 well plates as in example 1 scaffolds (β -TCP, 1% Sn @ β -TCP, 3% Sn @ β -TCP, 5% Sn @ β -TCP scaffolds) were placed in a scaffold (β -TCP, 1% Sn @ β -TCP, 3% Sn @ β -TCP, 5% Sn @ β -TCP scaffold), and bone marrow mesenchymal stem cells or human umbilical vein endothelial cells were seeded per well: 1 x 10^4 cells, adding the culture medium for total 500uL, culturing for total 48h, discarding the culture medium, washing twice with PBS, adding 500uL of culture solution containing 10% cck8 reagent into each well, reacting for 1h, taking out the supernatant, and measuring the absorption at 450nm in a 96-well plate by a microplate reader.

2. Staining of viable cells

Scaffolds (β -TCP, 1% Sn @ β -TCP, 3% Sn @ β -TCP, 5% Sn @ β -TCP scaffold) were placed into 48 well plates, and bone marrow mesenchymal stem cells or human umbilical vein endothelial cells were seeded per well: 1 x 10^4 cells, adding the culture medium for total 500uL, culturing for total 48h, discarding the culture medium, washing twice with PBS, adding 5uL CFSE dye into each well, staining for 20min, and taking an image by using an inverted fluorescence microscope.

As shown in FIGS. 4a and b, absorbance values at 450nm are detected by a cell proliferation detection kit (CCK8), and survival conditions of bone marrow mesenchymal stem cells (BMSC) and Human Umbilical Vein Endothelial Cells (HUVEC) on a beta-TCP, 1% Sn @ beta-TCP, 3% Sn @ beta-TCP and 5% Sn @ beta-TCP scaffold for 48h are examined, so that the absorbance of the scaffold (1% Sn @ beta-TCP, 3% Sn @ beta-TCP and 5% Sn @ beta-TCP scaffold) with different tin doping ratios and the absorbance of the scaffold (1% Sn @ beta-TCP, 3% Sn @ beta-TCP and 5% Sn @ beta-TCP scaffold) without tin doping at 450nm are close, and the biocompatibility of the tin-doped TCP scaffold is good. As shown in fig. 4, live cell staining of cells on the scaffolds with a commercial live cell dye (CFSE) also suggests that tin-doped β -TCP scaffolds also have good biocompatibility compared to non-tin-doped β -TCP scaffolds.

Example 5: influence of Sn @ TCP scaffold on osteogenic differentiation capacity of BMSC cells

A β -TCP, 1% Sn @ β -TCP, 3% Sn @ β -TCP, 5% Sn @ β -TCP scaffold was prepared as in example 1. Scaffolds (β -TCP, β -TCP @ 1% Sn, β -TCP @ 3% Sn, β -TCP @ 5% Sn scaffolds) were placed in 24-well plates, and each scaffold was seeded with BMSC cells: 3 x 10^5 cells, adding the culture medium for total 500uL, co-culturing for 14d, removing the culture medium, washing twice with PBS, dyeing by using a BCIP/NBT alkaline phosphatase color development kit, and taking a picture by using a hand-held magnifier.

As shown in figure 5, the scaffold is dyed by a BCIP/NBT alkaline phosphatase chromogenic kit, the content of an osteogenic differentiation marker alkaline phosphatase is investigated, the influence of different scaffolds on the osteogenic differentiation capacity of BMSCs is judged, and the doping of Sn can enhance the osteogenic differentiation capacity of the TCP scaffold on the BMSCs. With the increase of the doping ratio of Sn, the capability of inducing BMSC to differentiate osteogenically is improved.

Example 6: influence of Sn @ TCP scaffold on BMSC cell gene expression profile

A β -TCP, 5% Sn @ β -TCP scaffold was prepared as in example 1. Scaffolds (β -TCP, 5% Sn @ β -TCP scaffold) and blank controls were placed in 24-well plates, and BMSC cells were seeded per well: 3 x 10^5 cells, adding the culture medium for total 500uL, co-culturing for 14d, removing the culture medium, washing twice with PBS, adding pancreatin for digestion, centrifuging, removing the PBS, adding a proper amount of Trizol lysate, repeatedly blowing and uniformly mixing until clear and transparent liquid is formed, transferring the clear and transparent liquid into an enzyme-free tube, and storing the clear and transparent liquid in a refrigerator at the temperature of-80 ℃. The samples were placed in dry ice and sent to behamike bio-inc for transcriptome sequencing. The analysis results were analyzed using the bioinformatics analysis flow provided by the BMKCloud (www.biocloud.net) platform of the BMKCloud of the bmeker cloud.

Differential expression screening analysis was performed on the samples. As can be seen by the Volcano Plot (Volcano Plot) of fig. 6A, TCP and 5% Sn @ β -TCP scaffold caused 966 differential BMSC cell gene expression compared to the blank control group, with 837 up-regulated genes accounting for 86.6% and 129 down-regulated genes accounting for 13.4%. In FIG. 6B, we plotted each set of differential genes as required, and found that there were 91 unique differential genes of the 5% Sn @ beta-TCP scaffold. Wherein, compared with a control group, the mitogen-activated protein kinase signal pathway closely related to cell proliferation and differentiation shows that a plurality of genes are obviously up-regulated in a 5% Sn @ beta-TCP group: MAPKAPK, TRAF2, TAB1, AKT, MKK3, JunD, NGF, Ras, MEK2, NF κ B. (FIG. 7)

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (4)

1. A bone repair scaffold characterized by: the tin-doped calcium phosphate is used as a main raw material and is formed by 3D printing, wherein the doping amount of tin is 1-5 wt%.

2. A method of making a bone repair scaffold according to claim 1, wherein: the method comprises the following steps:

(1) designing a support structure: the cylindrical shape has the bottom area of 8mm and the height of 4 mm; the Simplify3D software parameters are set as: the printing speed is 4mm/s, and the filling rate is 40 percent;

(2) preparation of printing ink: sieving the beta-TCP powder by a 200-mesh sieve, and uniformly mixing the beta-TCP powder with tin powder to obtain Sn @ beta-TCP mixed powder with the metal tin content of 1-5%; adding the frozen section embedding agent according to the mass ratio of 1:1, and mixing and stirring for 10min to obtain printing ink;

(3) and printing the printing ink into a support through an extrusion type 3D printer, drying at room temperature for 48h, and putting the support into a muffle furnace for high-temperature sintering to obtain the bone repair support.

3. The method of claim 2, wherein: the frozen section embedding medium is aqueous solution of polyethylene glycol and polyvinyl alcohol.

4. The method of claim 2, wherein: the high-temperature sintering is carried out at a heating speed of 10 ℃/min, and the temperature is raised to 700-.

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